Introduction
Metabolic Solutions offers project design assistance
and a mass spectrometry service to help researchers
study lipid metabolism using stable isotope methods.
Stored triglycerides in the body can be mobilized
from fat cells. The process of triglyceride
breakdown or lipolysis results in the release
of fatty acids and glycerol. Fatty acids can
serve as energy substrates while glycerol can act
as a gluconeogenic precursor. Isotopic tracers
(palmitate, glycerol) can be used to quantify the
rate of appearance of fatty acids and glycerol into
the blood stream.
List of Lipid Metabolism Services:
- Fatty acid oxidation / fatty acid turnover
- Glycerol kinetics to detect fatty acid triglyceride
cycling
- Endogenous synthesis of cholesterol
- Rates of lipogenesis
- Desaturation and elongation of fatty acids
- Ketone body metabolism
- Cholesterol absorption
Fatty Acid Turnover
A stable isotope labeled fatty acid,
typically 13C-palmitate, is continuously
infused intravenously in tracer amounts. The rate of
appearance of endogenous unlabeled fatty acids into
the bloodstream can be determined by calculating the
dilution of infused isotope. Upon reaching steady-state,
the rate of appearance equals the rate of disappearance
or uptake. Therefore, the rate of appearance is equal
to the flux or turnover rate of the substrate.
Glycerol Turnover
The rate of appearance of glycerol
is a direct index of lipolysis. Fatty acid flux
can underestimate the rate of lipolysis except under
fasting conditions because of reesterification.
Fatty acids can become reesterified within adipocytes
which prevents release of fatty acids into the bloodstream
despite active lipolysis. However, glycerol cannot
be reincorporated into triglycerides because glycerol
kinase is absent within adipocytes.
A stable isotope tracer of glycerol (typically, D5-glycerol)
is continuously infused. A priming dose of tracer
is used to achieve steady-state levels quickly.
The stable isotope approach is advantageous compared to
radioactive tracer methods because gas chromatography-mass
spectrometry (GC/MS) methods measure isotopic glycerol
directly. Specific activity of glycerol is difficult
to measure because glycerol must be isolated from glucose
before counting the radioactivity. GC/MS methods
can also be used to accurately measure blood concentrations
of glycerol in the same analysis.
Rates of Fatty Acid Futile Cycle
Lypolysis and subsequent reesterification
of released free fatty acid represent a futile cycle.
This futile cycle allows the adipocyte to rapidly adjust
free fatty acid levels in meeting energy demands.
Simultaneous isotopic infusions of labeled fatty acid
and glycerol tracers will provide an index of the relative
rate of fatty acid reesterification. Three fatty
acids are released per glycerol molecule released.
If triglycerides are hydrolyzed within adipocytes and
subsequently fatty acids are reesterified and do not
enter the blood stream, then this results in intracellular
recycling. The intracellular recycling will be
equal to 3 times the flux of glycerol minus the free
fatty acid flux. Recycling can also occur when
free fatty acids are released into the bloodstream and
eventually reesterified. This would be classified
as extracellular recycling. Extracellular recycling
is calculated as the free fatty acid flux minus the
total fat oxidation. Therefore, total recycling
equals 3 times the glycerol flux minus total fat oxidation.
Fatty Acid Oxidation
The rate of fatty acid oxidation can be estimated by infusing
a C-13 fatty acids an measuring the rate of excretion
of expired 13CO2 in the breath.
The procedure requires the obtainment of a steady-state
level of 13C-fatty acid in the bloodstream
and in expired 13C-labeled carbon dioxide.
Using priming doses of sodium bicarbonate before the continuous
infusion of tracers will allow isotopic equilibrium by
60 minutes.
Protocol
Best Tracers: 1-13C-Palmitate
and D5-Glycerol
Priming Doses: D5-Glycerol
(1.5 µmol/kg/min)
1-13C-Palmitate (none)
Sodium Bicarbonate (0.07 mg/kg)
Infusion Pump Speed:
0.174 cc/min
Infusion Rate: Glycerol
(0.10 µmol/kg/min)
Palmitate (0.04 µmol/kg/min)
Sampling Times: 0, 60,
70, 80, 90 min (Plasma and Breath)
Diet Protocol: Fasted
or Fed
References: Wolfe et al.,
Biomedical Mass Spect. 7:168-171, 1980
Shaw and Wolfe, Ann. Surg. 205:368-376, 1987
Wolfe and Peters, Am. J. Physiol. 252:E218-E223, 1987
Klein et al., Am. J. Physiol. 257:E65-E73, 1989
Preparation of Tracer
Glycerol is infused as a sterile pyrogen-free
solution. Normal saline is used to dilute the
glycerol to the appropriate concentration. Before
infusion of palmitate, the tracer must be bound to albumin.
The palmitate-albumin mixture is prepared by first dissolving
a known quantity of palmitate in hexane, using sterile
containers. Use enough hexane to completely dissolve
the palmitate. An equimolar quanitity plus 3%
excess of KOH (dissolved in 80% mehanol) is added to
the hexane solution. The solution is evaporated
to dryness with nitrogen using a heated water bath (or
sand bath) at 60 °C. Preheated (60 °C) sterile
water is added to the dry potassium salt of palmitate.
Use enough water to solubilize the dry salt. Transfer
the aqueous solution with a heated (60 °C) sterile
syringe attaced to a Millipore™ (0.22 micron) filter
to a bottle of sterile human albumin (Cutter Laboratories,
Emmeryville, CA).
Calculations
Ra (µmol/kg/min)
= (Ei/Ep-1) x I
where Ra = rate of appearance of substrate, Ei
= Enrichment of infusate (atom % excess, APE), Ep
= Enricment of substrate in plasma (APE), and I = infusion
rate (µmol/kg/min).
FFA oxidation (µmol/kg/min)
= (Eb x VCO2 x 16)/(Ep
x k x %palmitate)
where Eb = enrichment of breath CO2,
VCO2 = µmol/kg/min Ventilation rate, Ep
= enrichment of palmitate in plasma, k = correction
factor for retention of bicarbonate in blood (0.81)
and % palmitate = the % palmitate concentration in blood.
Intracellular Recycling =
3 x Ra glycerol - Ra FFA
Extracellular Recycling =
Ra FFA - Total Fat Oxidation (indirect calorimetry)
Total Recycling = 3 x Ra glycerol
- Total Fat Oxidation
Published Lipid Metabolism Studies
Analyzed By Metabolic Solutions
1. Friedlander, A.L., Casazza, G.A.,
Horning, M.A., Buddinger, T.F., Brooks, G.A., Effects
of exercise intensity and training on lipid metabolism
in young women. Am. J. Physiol. 275(38):E853-E863, 1998.
"We examined the effects of exercise intensity
and training [12 wk, 5 days/wk, 1 h, 75% peak oxygen
consumption (VO2peak)] on lipolysis and plasma free
fatty acid (FFA) flux in women (n =8; 24.3 +/- 1.6yr)."
2. Cincotta, A.H., MacEachern, T.A., Meier, A.H. Bromocriptine
redirects metabolism and prevents seasonal onset of
obese hyperinsulinemic state in Syrian hamsters. Am.
J. Physiol. 264(27):E285-E293, 1993.
"Metabolic
and hormonal effects of bromocriptine were studied in
seasonally obese female Syrian hamsters. Lipid
mobilization (rate of glycerol appearance) was measured
after 10 wks of treatment."
3. Wray-Cahen, D., Caperna, T.J., Steele, N.C. Methyl-beta-cyclodextrin:
an alternative carrier for intravenous infusion of palmitate
during tracer studies in swine (Sus scrofa domestica).
Comp. Biochem. Physiol. A Mol. Integr. Physiol. 130(1):55-65,
2001.
Fatty acid-free albumin has been the standard carrier
for intravenous infusion of fatty acids to study in
vivo lipid metabolism. However, subjects can have
adverse reactions to infusion of albumin. We sought
an alternative to albumin as a carrier for intravenous
infusion of fatty acids, using the pig as a model.
13C-palmitate-methyl-beta-cyclodextrin was
infused under fasted and fed conditions in 50-kg pigs."
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